High resolution DNA melting assays for detection of Rx1 and Rx2 for high-throughput marker-assisted selection for extreme resistance to potato virus X in tetraploid potato
Nie, X., Dickison, V.L., Brooks, S., Nie, B., Singh, M., De Koeyer, D.L., Murphy, A.M. (2018). High resolution DNA melting assays for detection of Rx1 and Rx2 for high-throughput marker-assisted selection for extreme resistance to potato virus X in tetraploid potato. Plant Disease, [online] 102(2), 382-390. http://dx.doi.org/10.1094/PDIS-07-17-0968-RE
Plain language summary
Potato virus X (PVX) is a common and important virus of potato worldwide, causing economic losses in susceptible potato varieties. Since there is no cure once a plant is infected with a virus, PVX is mainly managed by using resistant varieties and virus-free seed potatoes. Therefore, breeding potato varieties with resistance to PVX is important in Agriculture and Agri-Food Canada’s potato breeding program and in other potato breeding programs around the world. Developing PVX-resistant potato varieties still involves inoculating the plants with PVX to determine which are resistant, a labor-intensive and inefficient process called phenotyping. In this research, we developed a molecular-based method for efficient and accurate screening of potato plants for two PVX-resistance genes, Rx1 and Rx2. We evaluated the new method by screening 42 potato varieties already known to be resistant or susceptible to PVX as well as 600 offspring of 12 crosses whose reactions to PVY were unknown, and demonstrated the method to be highly efficient and accurate. Potato plants which tested positive for Rx1 exhibited resistance to PVX, whereas those that tested negative for Rx1 were susceptible to PVX infection. This work shows that the new method is suitable for testing large numbers of potato crosses rapidly, reducing labour and time required to develop new PVX-resistance potato varieties.
Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/ 5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 were also performed. The efficacy of theHRMassays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.